1. Field of the Invention
This invention relates to the use of oil-in-water submicron emulsions as vaccine adjuvants for enhancing the immunogenicity and improvement of the immune response of antigens and to methods and compositions for preparing them. The invention further relates to a novel vaccine delivery system using proteosomes hydrophobically complexed to lipopolysaccharides and formulated for oral or intranasal administration to induce protective antibodies in sera and/or respiratory and/or intestinal secretions that are associated with protection against disease.
2. Background of the Invention
In the past, the risks of whole-pathogen vaccines and limited supplies of useful antigens posed barriers to development of practical vaccines. Today, the tremendous advances of genetic engineering and the ability to obtain many synthetic recombinant protein antigens derived from parasites, viruses, and bacteria has revolutionized the development of new generation vaccines.
Although the new, small synthetic antigens offer advantages in the selection of antigenic epitopes and safety, a general drawback of small antigens is poor immunogenicity, resulting in low antibody titers and the need for repeated immunizations. This lack of immunogenicity has created an acute need to identify pharmaceutically acceptable delivery systems or adjuvants for these new antigens.
To improve the immune response antigens are usually mixed with adjuvant substances that stimulate immunogenicity. Immunological adjuvants have generally been divided into two basic types: aluminum salts and oil emulsions.
Aluminum phosphate and hydroxide (alum) have a long history of use as adjuvants. They are the only ones recognized as safe for this use by the Food and Drug Administration. Antibody levels against antigens in alum-based vaccines are clearly, although moderately, elevated above those obtained with the corresponding aqueous vaccine. However, aluminum compounds have not always enhanced the immunogenicity of vaccines, and the problem of inconsistent antibody production has been frequently cited. Occasional production of sterile abscesses and persistent nodules were also reported with alum-adjuvanted vaccines. Regarding long term side effects, researchers have suggested a link between aluminum and diseases of the brain, including Alzheimer""s disease (Edelman, R.: Vaccine adjuvants. Rev. lnf. Dis. 1980; 2:370-383).
The development of emulsified oil adjuvants emerged historically from the studies of J. Freund who observed a remarkable increase in both the antibody and delayed hypersensitivity response to killed mycobacteria if the organisms were incorporated in paraffin oil. There are two types of Freund""s mineral-oil adjuvants: Incomplete Freund""s Adjuvant (IFA), consisting of an approximately 50:50 water-in-oil emulsion, and complete Freund""s adjuvant (CFA), a similar preparation with inclusion of killed mycobacteria. The powerful antibody-stimulating effect of CFA has not been surpassed by any other adjuvant. However, because of severe pain, abscess formation, fever and granulomatous inflammation, CFA can be used only for experimental purposes and not in human or veterinary vaccines. The toxic reactions reported using mineral oil-adjuvanted vaccines were attributed to impurities in Arlacel A (principally mannide monooleate), the emulsifying agent used in the preparations.
The use of IFA in humans has been limited to those clinical situations in which aqueous vaccines are relatively impotent and aluminum compounds have not provided enough adjuvant activity. J. Salk made practical the use of IFA in human vaccines by using a highly refined mineral oil and a purified Arlacel A emulsifier free of toxic substances injected intramuscularly in thousands of recipients. However, occasional failure of IFA vaccines reported in humans, and the discovery that Arlacel A was carcinogenic in mice, despite the absence of increased tumor formation in humans, has restricted the use of IFA vaccine formulations.
Since CFA was the first successful adjuvant, most investigators followed the example of CFA in assuming that substitutes for each of the three components, viz. oil, emulsifier and immunostimulant, are necessary for formulating a successful adjuvant.
U.S. Pat. No. 4,772,466 and 4,606,918 disclose methods for enhancing the immunogencity of an antigen by emulsifying it with a polyoxypropylene-polyoxyethylene block polymer, a glycol ether-based surfactant, a metabolizable non-toxic oil, and an immunopotentiating amount of an immunostimulating glycopeptide.
Pharmaceutical compositions comprising an oil-in-water micron size emulsion, refined detoxified endotoxin, cell wall skeleton and trehalose dimycolate have been disclosed as vaccine adjuvants (U.S. Pat. No. 4,505,900 and 4,803,070).
International patent application (PCT) WO 90/14837 discloses adjuvant composition comprising a metabolizable oil and emulsifying agent in the form of an oil-in-water emulsion, where the antigen is added externally to the prepared emulsion (extrinsic formulation). All the examples in the disclosure contained the immunostimulating agent, MTP-PE, a lipophilic muramyl peptide derivative.
Shigella flexneri and other Shigella species present another unique challenge as a disease vector. Shigella represent a major cause of diarrheal diseases in developing countries (Keusch, G. T. and M. L. Bennish. 1991. in Evans AS and Brachman PS ed. Bacterial Infection of Human 2nd ed. New York and London: Plenum Medical p. 593.). It has been shown that type specific protection against shigellosis can be acquired in man after infection with a wild type or attenuated bacteria (Cohen, D. et al. 1988. J. Infec. Dis. 157:1068.; Herrington, D. A. et al. 1990. Vaccine. 8:353; Black, R. E. et al. 1987. J. Infect. Dis. 155:1260.) and there is direct evidence that anti-type-specific LPS antibodies are associated with this protection (Cohen, D. et al. 1988. J. Infec. Dis. 157:1068.; Black, R. E. et al. 1987. J. Infect. Dis. 155:1260). It is widely agreed that local mucosal immune responses, especially secretory immunoglobulins including IgA and IgG play a major role in protection against such mucosal enteric pathogens following mucosal immunization or natural exposure; serum levels of these antibodies may be a measure or marker reflecting the production of local antibodies and, as such, may also indicate or contribute to protection (Underdown, B. J. and J. M. Schiff. 1986. Ann. Rev. Immunol. 4:389-417; Cohen, D. et al. 1988. J. Infec. Dis. 157:1068.).
Since the demonstration in 1967 (Formal, S. B. et al. 1967. Proc. Soc. Exp. Biol. Med. 25:347-349) that parenteral immunization with live or killed shigella bacteria was ineffective in protecting against oral challenge or monkeys with shigella, the major thrust of research has focused on the use of live attenuated or genetically constructed vaccines (Formal, S. B. and M. M. Levine. in Bacterial Vaccines, pp. 167-186). The problems associated with development of successful live vaccines include the narrow window between efficacy and safety of such vaccines since their ability to cause disease and side effects can be exceedingly dose dependent. The novelty of the current approach is emphasized by the fact that results of immunogenicity and protection in established animal models of disease were achieved despite using a sub-unit, non-living vaccine delivery system that is safe for intranasal or oral delivery. While several other approaches to the problem of development of oral or intranasal vaccines to protect against mucosal diseases in the gastrointestinal or respiratory tract have been explored, none uses the technology of the instant invention; nor have they been effective in demonstrating induction of high levels of IgA and IgG in both mucosal secretions and sera as well as protection in established animal models as is here shown.
Proteosomes have previously been used with peptides (U.S. patent application No. 07/642,093 filed Jan. 16, 1991 which is a Continuation of Ser. No. 07/065,440 filed Jun 23, 1987) and large proteins (US patent application entitled xe2x80x9cImmunopotentiating System for Large Proteins and Polypeptidesxe2x80x9d Ser. No. 07/336,952, filed Apr. 12, 1989) in vaccine development of parenteral vaccines and Zollinger et al. (U.S. Pat. No. 4,707,543; Nov. 17, 1987) have used meningococcal outer membrane proteins non-covalently complexed to detoxified lipopolysaccharides or polysaccharides in parenteral vaccines. The Zollinger work, however, specifically teaches away from the instant invention since the thrust of their work emphasizes that detoxified LPS or polysaccharide can be used whereas in the instant invention, detoxified LPS, in direct contrast to the non-detoxified LPS, is entirely ineffective. Furthermore, Zollinger neither showed, claimed nor suggested that his vaccines would be effective when delivered via the oral or intranasal route.
The present invention provides emulsions comprising a plurality of submicron oil-in-water droplets of a particle size in the range of 50 nm to 500 nm that effect enhanced immunogenicity of antigens incorporated intrinsically or extrinsically into the particles. Therefore the submicron emulsion (SME) particles of the present invention can be used as vaccine adjuvants.
In marked contrast to the aforementioned disclosures, as will be described, the present invention does not require use of any immunostimulatory mycobacteria or muramyl peptide-like additives for its submicron emulsion to be effective. Moreover, as will be seen, a preferred embodiment of the present invention consists of intrinsically incorporating the antigen into the emulsion at the time of formation of the emulsion; this is in distinct contrast to mixing the antigen with the emulsion after the emulsion has been independently extrinsically formed. It will be appreciated that intrinsic formulation will be effective even in situations and conditions and species where extrinsic formulation is not. In this regard as well, the present invention is uniquely different and not at all implied by the previously mentioned applications which indeed teaches away from the present invention in stating that it is sufficient to simply mix the antigen with the extrinsically previously formed emulsion.
The vaccine formulations of this invention also do not include any polyoxypropylene-polyoxyethylene block polymer, trehalose dimycolate, or cell wall skeleton, as are found in prior art compositions.
Another aspect of this invention is to provide compositions and methods for the preparation of submicron emulsions containing antigens, incorporated either intrinsically (emulsified together with the oil and surfactant) or extrinsically (added externally to prepared SME).
In some cases, the submicron emulsion of the present invention can be administered in combination with other vaccine delivery systems, such as proteosomes, as indicated in the examples.
The size, concentration and specific formulation of SMEs may be varied to suit the particular antigen used. Moreover, such adjuvant preparations may enhance both humoral and cell-mediated immunity (CMI) as do Freund""s adjuvants. The SMEs here described have been developed for human use and since the oily droplets of the emulsions are of submicron size and contain no added pyrogenic moieties such as mycobacteria or MDP derivatives they have, unlike Freund""s adjuvants, great safety potential. They may be especially applicable to antigens that are vaccine candidates to protect against biologic toxins or infectious agents which have natural hydrophobic moieties as a component including transmembrane viral, bacterial or parasite proteins, membrane proteins such as proteosomes, lipopolysaccharides, glycolipids such as gangliosides, or a variety of proteins or peptides to which hydrophobic anchors have been chemically or genetically added.
Another aspect of the invention provides compositions and methods to achieve mucosal immunity by using an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, immunogenic peptide or antigen, and an aqueous continuous phase, which induces mucosal immunity by achieving mucoadhesion of the emulsion particles to mucosal surfaces. Mucous surfaces suitable for application of the emulsions of the present invention may include ocular (corneal, conjunctival), oral (buccal, sublingual), nasal, vaginal and rectal routes of administration.
The emulsion particles have a hydrophobic core comprising a lipid or lipid-like composition and are stabilized with amphiphilic and/or non-ionic surfactants.
A wide variety of immunogens, including both water-soluble and water-insoluble peptides or polysaccharides, may be accommodated in the present emulsions. The hydrophobic core and surfactant provide a microenvironment which accommodates lipophilic immunogens such as lipid A or lipopolysaccharides as well as membrane-associated peptide antigen domains, while the aqueous continuous phase accommodates water-soluble peptide domains, or oligosaccharides.
The term xe2x80x9cpeptidexe2x80x9d herein includes both oligopeptides and proteins. To facilitate intestinal uptake, the emulsions may be encapsulated in gelatin capsules or otherwise enterocoated to prevent their exposure to gastric fluids when the oral route of administration is selected. Furthermore, the emulsions may be lyophilized as disclosed previously (Pharmos, PCT/US 93 01415) prior to their encapsulation in order to achieve added stability of the antigen.
Another invention is a desirable vaccine using lipopolysaccharide (LPS), e.g. Shigella flexneri 2a, Shigella sonnei or other shigella lipopolysaccharide (LPS), complexed with proteosomes to induce anti-LPS antibodies in the aforementioned fluids in the absence of SME particles which protects against homologous shigella infection in a well-known animal model of shigellosis. The data disclosed herein shows that the instant invention can be used as an oral or intranasal non-living sub-unit vaccine to protect against mucosal diseases of the gastrointestinal tract such as shigellosis. In addition, since high antibody levels are induced in either the respiratory or gastrointestinal tracts following either oral or intranasal immunization, and since protection is shown against either conjunctival or respiratory challenge, these proteosome-based vaccines and there associated methodologies can also be used to protect against diseases that enter the body via respiratory, ocular or gastro-intestinal routes. These vaccines should also result in protection against mucosal diseases of the urogenital and auditory tracts.